Automatic chemical analyzer



Feb. 13, 1968 s. NATELSON 3,368,872

AUTOMATIC CHEMICAL ANALYZER Original Filed Aug. 31, 1964 4 Sheets-Sheet1 m T m V m ww mm m 23x0 OF PIG] 55E 20530 H mm W& MU

ATTORNEY Feb. 13, 1968 NATELSQN 3,368,872

AUTOMATIC CHEMICAL ANALYZER Original Filed Aug. 31, 1964 4Sheets-Sheet'Z SAMUE L NATELSON INVENTOR ATTORNEY Feb 13, 1968 s.NATELSON 3,368,872

AUTOMATIC CHEMICAL ANALYZER Original Filed Aug. 31, 1964 4 Sheets-Sheet5 TRANSFER ZONE SAMUEL NATELSON INVENTOR.

ATTORNEY Feb. 13, 1968 s. NATELSON 3,368,872

AUTOMATIC CHEMICAL ANALYZER Original Filed Aug. 31, 1964 4 Sheets-Sheet4 109 I STANDARD STANDARD POROUS TAPE TEST TAPE SAMUEL NATELSONINVENTOR.

ATTORNEY United States Patent 3,368,872 AUTOMATIC CHEMICAL ANALYZERSamuel Natelson, Valley Stream, N.Y., assignor to Scientific Industries,Inc., Queens Village, N.Y. Original application Aug. 31, 1964, Ser. No.393,096, now Patent No. 3,260,413, dated July 12, 1966. Divided and thisapplication Feb. 14, 1966, Ser. No. 547,110

4 Claims. (Cl. 23-253) ABSTRACT OF THE DISCLOSURE A device for thechemical analysis of fluids automatically comprising a device fordispensing a liquid sample in the form of a drop on a tape combination,said tape combination comprising a transparent porous portion travellingwith and in close contact with an absorbent tape, said absorbent tapehaving been impregnated with a reagent for producing a color reactionwith the component of the sample sought, a transfer zone maintained atconstant temperature through which the drop of sample supported on saidporous tape passes and in which zone said drop and said tapes moves fora fixed period of time, the component for assay passing through saidporous tape to said reagent tape, a washing station where water flowsover the porous tape to remove the spent sample, a drying zone wherewarm dry air dries the tape combination and a readout zone where a beamof collimated monochromatic light passes through said tape combinationand is recorded by means of a photocell attached to a recorder.

The present application is a division filed under Rule 147 of Ser. No.393,096 filed on Aug. 31, 1964, now Patent No. 3,260,413, which is acontinuation-in-part of U.S. patent application Ser. No. 234,019 filedOct. 30, 1962 and now Patent No. 3,219,416, U.S. patent application Ser.No. 216,845 filed Aug. 14, 1962 and now Patent No. 3,261,686, U.S.patent application Ser. No. 170,084 filed Jan. 31, 1962 and now PatentNo. 3,216,804. The foregoing applications in turn relate back to U.S.patent application No. 14,894, filed Mar. 14, 1960, now U.S. Patent No.3,036,893 and, Ser. No. 216,845 relates back to U.S. patent applicationNo. 155,882 now abandoned.

This invention relates to the chemical analysis of substances, and moreparticularly to a method and apparatus for the automatic continuousanalysis of substances.

The chemical laboratory is often faced with the problem of the analysisof large numbers of samples. This is time consuming and tedious to theoperator, often resulting in error due to the large number of samplesbeing assayed and the close attention one must pay to each step of theprocedure. The fatigue which sets in often results in errors beingintroduced. In many cases, several operators are required to finish thework of the day.

To relieve this condition, instruments have been introduced whichautomatically sample the material to be analyzed, add reagents, and gothrough elaborate processes both as regards procedure andinstrumentation to perform the analyses automatically. These devices arecumbersome, elaborate and are expensive to produce. Thus, only certainlaboratories can afford the expense of these instruments. In addition,the operator must be skilled in handling the equipment as regardssetting up the instrument and adjusting it when something goes wrong.These instruments require appreciable amounts of material for analysisand do not lend themselves readily to adaptation for the analysis ofultramicro samples of the order of microliters. Furthermore, in theseinstruments, the sample and reagents are pumped in the instrument inliquid form so that an appreciable quantity of sample is needed.

In the aforementioned U.S. Patent No. 3,036,893, it

3,368,872 Patented Feb. 13, 1968 has been shown that small quantities ofsamples could be processed, treated and analyzed using, fiat mediumssuch as paper tapes. The present invention is closely related to thebasic concept of U.S. Patent No. 3,036,893 but includes simplifiedchemical analysis procedures.

It is therefore an object of the present invention to provide means forchemical analysis using flat mediums.

And, it is also an object of the present invention to provide a devicewhich does not require liquid reagents.

Another object of the invention is to provide a device which is easy tooperate.

Still another object of the invention is to provide a device whichpermits multiple analysis of several constituents simultaneously.

This invention also contemplates providing a device which is inexpensiveto manufacture.

It is also the purpose of the invention to provide a system which canhandle ultramicro samples.

The invention likewise contemplates providing a device which is rapidand capable of turning out a result in as sort a time as twenty secondsor even less, once the instrument has started to run.

Yet another object of the invention is to provide a device which isversatile, without changing to elaborate equipment during intermediatestages of the operation.

Furthermore, the invention contemplates providing a device which isreliable, having a minimum number of easily repairable parts.

Among the further objects of the present invention is the provision of adevice which can do highly colored solutions, e.g., hemoglobin, when thecolor does not readily pass through a membrane.

It is contemplated that the system herein described can be used forsingle and multiple determinations.

With the foregoing and other objects in view, the invention resides inthe novel arrangement and combination of parts, in the details ofconstruction, and in the process steps hereinafter described andclaimed, it being understood that changes in the precise embodiment ofthe invention herein disclosed may be made within the scope of what isclaimed without departing from the spirit of the invention.

The invention will appear more clearly from the following detaileddescription when taken in connection with the accompanying drawing,showing by way of example, preferred embodiments of the inventive idea.

FIGURE 1 is a longitudinal schematic view of one embodiment of theapparatus contemplated herein;

FIGURE 2 is a perspective view of an embodiment of a sample dispenserwhich may advantageously be used with the other apparatus hereindescribed;

FIGURES 2a, 2b, and 2c are illustrations of the operation of a portionof the sample dispenser shown in FIG- URE 2 but at diflerent timeperiods;

FIGURE 3 is 'a longitudinal schematic view of another embodiment of thepresent invention;

FIGURE 3a presents a portion of a longitudinal schematic view of amodification of the embodiment illustrated in FIGURE 3;

FIGURE 4 depicts an embodiment of the present invention useful forsingle determinations;

FIGURE 5 illustrates another embodiment of the present invention usefulfor a single determination; and

FIGURES 6a, 6b, 6c, 6d illustrate the use of the embodiment of FIGURE 5in making a single determination.

The invention in its broadest aspects contemplates a system for thetreatment of microqu'antities of samples, e.g., drops or droplets ofblood, urine and other body fluids so that quantitative and qualitativedeterminations can be made, and ailments can be diagnosed therefrom.

Generally speaking, the present invention provides for a flat testmedium or tape to which the sample will be transferred and upon whichtape the sample or samples will be subjected to treatment for analysis;and, a porous flat medium or tape through which the sample to beanalyzed will have to pass in order to reach the test tape. The poroustape may be bonded to the test tape by means of pressure or by sprayinga solution containing a plastic such as cellulose acetate or cellulosenitrate and allowing the solution to evaporate to form a thin porousfilm on the test tape.

When the porous tape is bonded to the test tape to form a combined tape,the sample may be analyzed by means of an apparatus having an input zonewhere the sample is placed on the porous portion of the bonded tape indrop quantities; a transfer zone 12 where the sample is transferredthrough the porous portion of the tape, to the test portion preferably'at constant temperature, a treating zone 13 where the combined tape issubjected to treatment such as applying heat to develop a color or stopan enzymatic reaction, or where reagents may be sprayed on the test tapeto develop a color; for analyzing the sample, and may include a Washstation 14a to remove excess sample and/or excess reagent and a dryingstation 14b a reading zone 15 where the treated sample is read, and aterminal zone 16 which includes means to take up the combined tape 18,e.g., 'a take-up reel. Driving means are provided to drive the tapeseither at a constant rate or intermittently.

The reason for using a plural tape arrangement will become apparent inconnection with the explanation of the operation of the device givenafter the following detailed description of the apparatus.

The samples to be tested or analyzed are contained in a dispenser 17 apreferred embodiment of which is depicted in FIGURE 2 of the drawing.The principal requirement of the dispenser is that it can either drop orblow small measured quantities of the sample at a desired place on theapparatus.

In accordance with one embodiment of the invention, the sample from thedispenser 17 (shown in FIG. 2) will be received on combined tape 18which preferably will have been treated with a reagent. Combined tape 18will be contained on a payoff, e.g., tape payoff roller 20. Forconvenience, no supporting means are shown for any of the numerousrollers or reels herein described, it being of course well understoodthat such supporting means are provided.

To receive the sample, there is provided a sample receiving station 28,having guide means 29, and it is towards this sample receiving station28 that the sample from dispenser 17 will be dropped. In order to avoidany contamination of the sample which will be placed on the samplereceiving tape, a horseshoe shaped recess may be provided in thesampling plate at the spot where the sample is placed on the tape. Thus,in this embodiment, the plate serves only as a support, and the sampleitself never touches the plate. To further insure accuracy in theplacing of the sample on the tape, idler roller 31 may be provided. Thiswill prevent the tape from having any crimps or bumps and aids in theuniformity of the sample spot placed on the tape.

In the embodiment shown in FIGURE 1, the combined tape 18 travelscontinuously from the input zone towards the terminal zone. After thesample spot has been placed on combined tape 18 and the tape hastraveled through the transfer zone at least some of the sample will havepassed to the test portion of the tape. The tape then enters thetreating zone 13. Here the sample may be heated to accelerate a chemicalreaction or stop an enzymatic reaction. In some cases an additionalreagent may be sprayed on to the test tape to develop a color. Excesssample and/0r reagent can be washed off at a wash station 14a and thetape may be dried at a drying station 1412. The tape is read by passinga thin pencil of light through the spot in the reading zone 15, andfinally is wound on a reel at the terminal zone. Instead of the sin glecombined tape arrangement of FIGURE 1, it is advantageous for somepurposes to use a separate test tape 21 and porous medium 23. The twotapes may be made to adhere by simple pressure and then wound on thesame pay-off 20a. The test tape 21 may contain a reagent. With regard toporous medium 23, the degree of porosity will depend on the sampletested and on the treatment to which the sample is subjected. Forproteins, total lipids, mucoproteins etc. where larger molecules arebeing assayed, the porosity of the tape must be increased to allow thesemolecules to go through. Likewise, the degree of porosity of the tapemay also be used as an effective means for separating components of thesamples being tested. In this connection, a porous tape made ofcellophane, cellulose acetate or cellulose nitrate has been foundparticularly useful for certain hospital laboratory ananlysis, e.g.,sugar, urea, calcium, uric acid, creatininc, and amino acids where it isdesired to prevent proteins from reaching the test tape.

After leaving the pay-off 20a, the tapes pass to the input zone wherethe sample is deposited on the porous tape.

These two tapes, i.e., the porous tape 23, and the test tape 21 are alldirected towards a pair of spring loaded rollers 27 located at theentrance of the transfer zone 12. The function of these rollers is tobring the tapes into close contact so that the sample can be transferredto the test tape through the porous tape. Just as the sample receivingplate 28 recesses so as to avoid contamination of the sample, so do thespring loaded rollers 27 likewise have a recess. The sample passesbetween the recesses.

The tapes now enter a treating zone 13 which is completely enclosed inenclosure 33, preferably made of transparent plastic, and advantageouslyhaving a door 34. This door is to facilitate adjustment or repairs andcan be a simple affair held by hinges 34b. Naturally the entireenclosure need not be transparent, just so long as the operator can seewhat goes on inside the enclosure and make certain that the device isfunctioning properly. The treating zone illustrated in the drawingcomprises a chamber 33 in which there is provided heating means 34a.Advantageously, a thermometer 35 may also be provided in chamber 33. Thechamber is preferably transparent, or partially so. To increase thetravel time in this zone, the construction of the chamber may beelongated and may have one or more idlers to change the course of travelof the test tape through the zone. Additional equipment may also beprovided in this zone, either in the chamber as shown in the drawing,such as a fan 37 and air vents all of which of course may be adjustable.A sprayer may be provided for spraying a reagent on to the test tape. Indesigning the equipment, the idler rollers 36 in the zone may beremovable and so disposed that the addition or removal of these rollersin the zone fixes the travel time in the zone each particulardisposition of these rollers in the zone causing the tape to travel inthe zone a predetermined time period. It has also been foundparticularly useful to have a washing station in the zone adapted towash the excess reagent or sample from the test tape. The test tape 21leaves the treating zone along an idler 39 at zone exit 40. The testtape now enters the reading zone 15 where the results of the test oranalysis will be read on a densitometer 41. An appropriate light filter42 acting in combination with a light source 43 is on one side of thetape, and a photo cell 44 is on the other side. The action of the lighton the densitometer will be plotted on graph paper which shows the peaksmade by a pen. This type of densitometer is known in the art.

At the treating zone exit another idler 51 is provided to facilitate theseparation of the tapes. Here the porous tape is separated from the testtape. Porous tape takeup reel 52 is provided for this purpose and thetapes are moved by a motor or belting arrangement 53. In this manner thesample remaining is removed and discarded.

The test tape has now served its purpose and has come to the end of itsroad. It therefore enters the terminal zone. The test tape finally windsup on takeup reel 16a shown in the drawing as being rotated by belting53a. The drive for the device should be such that by proper adjustment,it should be both continuously driving the rollers or intermittentlydriving the rollers. Usually, when the sample from dispenser 17 is beingdropped on the tape over the sample receiving plate, or even when blownor dropped thereon by the operator, the tapes are moving at theintermittent rate, allowing the sample to drop on the tape while thetape is not moving. However, after one or a series of samples have beendropped, the operator may wish to move the tape along for some distancebefore starting a new series of tests with other samples. It is thuspreferable for the operator to be able to advance the tape continuouslyby pressing a button or switch which will change the intermittent driveto continuous drive.

In the apparatus of FIG. 3, both the porous tape and the test tape arewound on the same pay-off. It is also possible to provide a moreflexible instrument shown in FIGURE 3a, the reagent being varied withthe procedure employed. Thus, the reagent can be changed instead ofchanging test tapes for each analysis. The porous tape 23a and the testtape 21a are wound on separate pay-offs 20b and 200. Guide rollers 22direct the two separate tapes. The test tape 21a passes over a reagentcontaining station 25 having a wick contact roller 25a. The tape movesover the roller 25a which turns. Since a reagent 24 is contained in thestation container, the wick contact roller 25a brings up the reagent tothe underside of the tape impregnating the tape with the reagent. Thetwo tapes are directed to a pair of spring loaded squeeze rollers 27aand thence to the input zone where a sample is deposited on the tapefrom a peristaltic pump. The test tape may be dried before it contactsthe porous tape by passing through a ventilated zone 25b at a controlledelevated temperature.

The apparatus of FIGURES 1, 1a, 3, 3a, can monitor the changingconcentration in a batch solution. In this case the sample is deliveredby a peristaltic pump which pumps specimens continually. These arrive atthe porous tape in the form of drops from a narrow nozzle. The drops arespaced about an inch apart on the porous tape.

The changes in the composition of the concentration of the drops aredetected at the reading station. The output from the photocell sensingmeans is recorded as peaks and, a continuous recording of the changingconcentration is thus recorded as a series of changing peaks.

As examples of batch solutions that have been used are fermentationmixtures, blood from a cannula inserted in the artery or vein of anexperimental animal or a human, plating baths, organic reactionsmixtures and the like.

In connection with the devices just described, it is advantageous tohave associated therewith a sample dispenser 17, preferably driven bythe same driving means as are used to drive the device. The dispensershould be removeable or detachable from the device to permit manuallyfeeding the sample receiving tape. The dispenser includes circularpositioning means '79 on which a plurality of radial holders 80 aremounted near the edge of the positioning means. In each holder 80, thereis provided a bed 82 made of rubber or other resilient material intowhich a capillary tube 83 is inserted radially into the holder. Thecapillary tubes 83 are held in the holders 80 by means of a threadedknurled fastener 81 which is spring loaded. The spring is released bymeans of release means 84 which projects radially beyond the peripheryof the positioning means '79. As the circular positioning means 79 turnsit brings the radially disposed capillary tube into the input zone.Here, the capillary tube 83 will be seized by a clamp arrangement 85'.Clamp arrangement includes lower and upper clamp jaws 86 and 87 normallydisposed radial to circular positioning means 79. Clamp jaws 86 and 87are pivotly mounted by a pivot 88 on a hinged arm 89. As the releasemeans 84 hits lower clamp jaw 86, the knurled fastener is released.Lower clamp jaw 86 moves up while upper clamp jaw 87 remains stationaryclamping the capillary tube 83 in between. As shown in FIGURES 2a and 2bthe capillary tube secured between the clamp jaws is first raised so asto clear the circular positioning means. The clamp holding the capillarythen swings 180 with the capillary tube to a point over the horseshoeshaped recess 30. Hinged arm 89 which is normally horizontally disposedthen turns on hinge 90 to the vertical position as shown in FIGURE 20discharging the contents of the capillary tube. The hinged arm 89 thenreturns to the horizontal position, and the clamp jaws 86 and 87 swingback to a horizontal position. The clamp and capillary now swing back 90to a position intermediate the tape and sample holders. The bottom clamplowers, releasing the capillary which falls into a waste container. Theopen clamp now moves another 90 to be in a position to pick up the nextcapillary. Circular positioning means capable of holding fiftycapillaries or more can be constructed. When the capillary tubes havebeen inserted in the horizontal position they can be rapidly filled bysimply touching the sample to the tip of the capillary. It then runsright to the other end of the tube. For most tests, capillary tubes ofbetween 0.2 to 0.5 mm. bore and capable of containing from 0.01 to 0.05ml. are preferable. But, in some cases, capillary tubes of less than 0.1mm. bore are used.

In practice, the operator places the capillary tubes on the circularpositioning means and tightens them in place with the knurled fasteners.He then touches the liquid samples to the capillaries which fill bycapillarity. When the capillaries turn 180 the end not touched to thesample eventually approaches the porous tape and deposits the sample.The capillaries need not empty but will deliver the same amount eachtime. The capillaries may be emptied by compressed air blowing on theupper end. In this manner the outside of the tube which holds somesample by adhesion, when the capillaries were filled, is not broughtnear the tapes. Thus accurate measurement is practicable withoutcontamination.

Where single determinations are required such as on a patient in aphysicians oflice, it is advantageous to use strips of tape prepared asindividual pads shown in FIG- URE 4. Here a plurality of flat mediums ontapes are shown as individual strips stapled together by a staple 101.The sample is placed on a sample receiving tape 102, then goes through aporous tape 103 onto a test tape 104 these tapes are retained sandwichfashion by staple 101.. The sample receiving tape is shown with aconfined spot circle 105 on the top of the tape although it may also beplaced on the bottom. The test tape which usually contains a reagent isencased in a solvent-repellant polyethylene bag 106 and the entireassembly is covered by a water-repellant cover 107. The operation ofthis pad can best be understood from a description of the operationperformed.

The patients finger is stuck in the conventional manner and a drop ofblood is taken up in a calibrated capillary or pipet such as a Sahlipipet. The outside tip of the pipet is wiped and the blood is touched tothe center of the paper of the sample receiving tape strip. The blooddrains on to the paper completely by capillarity and, a confined spot isadvantageously used for greater accuracy, although it is not necessaryfor approximate results. The porous intermediate tape layer is notwetted but the bottom test tape paper strip is moistened with a reagentor already has a reagent thereon. When determinations are made, it mayor may not be necessary to turn over the sample receiving tape strippaper. Very often, the sample will permeate right through the tape sothat the reversal of the tape strip is unnecessary. In fact, when greataccuracy is not required the sample may be deposited directly on theporous tape. The pores of the intermediate porous tape strip arecontrolled so that for determinations like sugar and urea. onlysubstances of low molecular weight can pass. For protein estimation, apore size is chosen so as to allow the proteins through but not allowthe red cells to pass. For the analysis of substances such as aqueoussolutions or human serum, the test tape may also be dry but impregnatedwith reagent. In this case, moisture is supplied from the sample. Forshipment, the reagent-containing test tape may be encased in apolyethylene container so that the solvent does not evaporate, i.e., thereagent-containing test tape strip is kept in a separate bag which istorn open just before use and inserted below the intermediate poroustape. When the twotape or three-tape strips are assembled and sold aspart of a pad, the bag is torn and removed just before use.

After the blood has been placed on the sample receiving or porous tapeor strip of the pad, FIGURE 4, the pad may be, for greater accuracy andsensitivity, placed in a pressing device which will press the tapes orstrips together. The pressing device may include heating means, and isdescribed in my co-pending application Serial No. 216,845 filed August14, 1962 as hereinbefore mentioned.

The pad assembly of FIGURE 4 may be combined into a single strip asshown in FIGURE 5. This strip consists of an elongated section ofabsorbent test paper 103 having a porous portion 169 over the tip of thetest paper. The porous portion 109 over the tip may be bonded thereto byspraying a solution containing a plastic such as cellulose acetate orcellulose nitrate and allowing the solution to evaporate to form a thinporous film on the test tape. The area under the porous portion 129 isimpregnated with a reagent. A water repellant medium 110 may be placedacross the tape and impregnating same to produce a confined spot asshown in FIGURES 6a to 6d. Placing a drop of sample on the porous tapewill cause some of the component being assayed to permeate to the testtape producing a spot suitable for visual comparison with a standardcolor chart or assay by means of a densitometer as described above forthe continuous procedures. The standard or standards, for visualcomparison may be provided right on the tapes next to a confined spot asshown in FIGURE 5.

An alternative method is to apply the sample on the porous tape whichhas been pressed on to the test tape so that it adheres but allowing aportion of the porous tape to extend beyond the test tape. After a dropof sample has been applied to the porous tape and a predetermined timehas been allowed for it to permeate the porous tape nad react with thetest tape, the porous tape may be lifted and discarded permitting thetest tape to be examined by visual comparison with a color chart or bymeans of the densitometer as described above for the continuousprocedures for multiple samples.

Although the foregoing explanation is suflicient to understand theoperation of the components of the embodiments herein contemplated, itis believed that additional explanation is required to clarify thereason for using the arrangements described, particularly thecombination of the tapes. As already explained at the beginning of thispatent specification, tape devices, particularly where paper tapes areemployed have often not been considered suitable for quantitativechemical analysis of substances. This is because of the irregular spotformation. Obviously, the reading means, e.g., densitometer has no wayof distinguishing between even and uneven spots. Furthermore, if thecomponents of the sample drop or specimen are not properly or uniformlydistributed, visual study of the curves and peaks recorded by thedensitorneter is impossible. If two spots containing the same componentsare distributed over an equal area but in a different manner, thedensitomcter recording device will record two different curves. Theareas of each curve will be precisely the same, but the peak height ofone curve will be different from the other. Electronically, both spotscan be read properly and correct results obtained, but visual comparisonis of course impossible.

The need for a plurality of tapes can be understood from the followingobservation. When a drop of water is dropped on a piece of paper whichhas been impregnated with a colored reagent which is water soluble, thewater will spread. In so doing, it will dissolve the reagent and move itaway from the center where the water has been dropped. On drying, a ringwill be observed. The center of the ring will be white, where thereagent has been washed away. The outer ring will be dark where thereagent has been concentrated. For this reason, it is impractical toperform a quantitative test by dropping a test solution on a reagentpaper. If attempted, one would obtain a thin ring surrounding the areaof application. When sent through a densitometer, the densitomer willread in error. The center of the spot will allow too much light to gothrough since it is clear. The ring concentration will be underestimatedsince light will not pass the high concentration of color in the ring.Thus, whether there is a certain amount or whether there is twice thatamount in the ring, the maximum absorption of light will have beenreached and the amount will be underestimated. When the sample isapplied through a porous tape with minute pores, submicroseopic dropletsof test solution are evenly distributed over the reagent paper. Thisresults in an even distribution of the sample on the reagent paper. Thecolored product of such a reaction, can be accurately estimated by thedensitometer.

If samples of different concentrations are applied through the poroustape then diffusion through the porous tape of the components dissolvedin the sample will be a function of their concentration. The higher theconcentration of the sample the higher in concentration will be thematerial being diffused. Thus only a portion of the de' sired componentneeds to be diffused to produce a stain on the reagent paper theintensity of which is a measure of the concentration of the substancesought. Thus, if the size of the sample drop placed on the porous tapevaries then a stain of varying area will be produced but the intensityof color will be the same for the same concentration.

If the sample resting on the porous tape is now removed by lifting ofthe porous tape or by wiping or rinsing the sample off with a solventsuch as water or alcohol. The concentration of substance in the originalsample can be estimated by visual comparison with results obtained fromsamples of known concentration. As an alternative a beam of lightsmaller than the area of the stain can be passed through the stain,evaluating its sensitivity by means of a photocell attached to a readingdevice such as an ammeter or recorder.

The need for confined spots for some determinations can be understoodfrom the following observations: When samples containing 20 ,ul. ofhuman blood serum and water are placed on a paper tape, the serum willspread normally to about 17 mm. and the water to about 20 mm. For thisreason, aqueous solutions cannot be compared with serum. The samequantity of serum or water can be confined in a confined spot circle ofabout 15 mm. diameter. The 15 mm. spot keeps them spread equally anddistributes their contents over an equal area. When the water contains ameasured amount of sugar, the sugar content of the serum can be comparedwith the sugar content of the water by comparing the intensity of thestain produced. The confining circles can be made of wax, polyethylene,glyptal resin, urea formaldehyde resin, C-oil polymer or other suitableplastic that is hydrophobic. Since the confined spot is smaller than thearea over which the sample would spread normally, the confined spot actsas a container for a liquid which when laced on the porous tape permitsthe diffusion of the desired substance through the porous tape to thereagent tape.

General procedure used Twenty microliters of the sample mentioned, e.g.,blood serum or blood were placed on the porous tape. This is done eitherby blowing the sample from an ultramicro pipet and touching it to thespot or :from an automatic device as shown in the drawing. Theinstrument is started and travels a short distance, about 1 /2 inchesand stops. A second sample is then blown or dropped on the tape. Thiscontinues until all the samples have been added. The instrumentcontinues to move until the tape strip emerges with the samples thereonwhich have been processed and spots are read.

Tape construction When separate tapes are used, the porous tape is madeof cellophane, cellulose acetate or cellulose nitrate of 0.1 to 1 mil inthickness. This is untreated and in this condition, permits smallermolecules such as water, sugar and salts to pass through, but notmacromolecules such as protein. To pass larger molecules such asproteins, porous tape may be perforated cellophane, polyethylene,Teflon, cellulose acetate or polypropylene or a closely woven nyloncloth. The bottom tape is made of paper 1 /2 inches wide. This may bebacked with a layer of polyethylene to give it -wet strength. Othertapes tested as the bottom test tape were paper tapes with cotton threadwoven in them and paper tapes impregnated with plastic on both edges ofthe paper to a distance of inch, leaving the center inch untouched. Allwere found satisfactory. A closely woven cloth made of cotton was alsotried and found satisfactory as the test tape.

Example I.-G1uc'0se in blood serum (enzymatically) To 200 ml. of -N/phosphate butter, pH 7, add 10 mg. of horseradish peroxidase and 250 m.of glucose oxidase. Shake until dissolved. Now add 1 ml. of a 16%O-dianisidine solution in water and mix. Filter this solution andimpregnate the test paper with it. Air dry the paper. Add 0.02 ml. ofhuman blood to the porous tape surface which is on top of the test tape,from the automatic feeding device of FIGURE 2 or from an ultramicropipet. The porous tape in this case is untreated cellophane, orcellulose acetate as shown in FIGURE 1. The transfer zone is maintainedat a temperature of 37 C. in this case. Contact time in the transferzone is two minutes. The tape is heated in the treating zone at 70 C. tostop the reaction. The sample is washed off the tape at the wash stationand then dried at the drying station if the bonded tape of FIGURE 1 isused. If the tapes are separate as in FIGURE 3, the washing and dryingis dispensed with and the top tape is lifted 01f thus removing thesample. The dried bonded tape or the test tape now passes thedensitometer so that the concentration of glucose in the sample can beevaluated. The filter used in the densitometer has a transmissionmaximum of 395 mg. The height of the peaks on the recording chart are ameasure of the glucose concentration.

Example Il.Glucose in blood serum (by chemical means) A 1% solution ofpotassium terricyanide is acidified to a pH of 4 with dilute phosphoricacid. The test paper is dipped in this soluxtion and air dried. Serum(0.02 ml.) is added to the tape and the procedure followed as in ExampleI, using cellophane (0.8 :mil) as the intermediate tape. Ditfusion timeis two minutes at 37. The filter used in the densitometer is a 540 mfilter. The drying oven in the treating zone in this case is heated at85 C. to develop a blue color. For this procedure as an alternative theinstrument of FIG URE 3a is used. In this case the test tape is allowedto remain moist (not dried) be- 10 fore it comes in contact with theporous tape. In this procedure the lower tape is heated at C. tocomplete the reaction and to dry the test tape after the porous tape hasbeen removed and before it is read in the densitometer.

Example [IL-Carbon dioxide in blood In this case a porous tape is usedwhich allows gases to go through but not proteins. Salts and water cango through but more slowly than the gases. Such a tape can be mostreadily made from porous Teflon (0.1 mil in thickness) and alsocellulose acetate. The instrument of FIGURE 3 may be used for thispurpose. In this case the lower tape is moistened with a solutioncontaining phenolphthalein with a 7 M phosphate buffer of pH 8.0. Thelower tape remains moist and is not dried. The sample in the form of alarge drop is placed on the porous tape which is touching and above thenet test tape. The carbon dioxide passes through the porous tape anddecreases the intensity of the pink phenolphthalein by acidification.The Teflon tape is lifted off the test tape and the decrease inintensity is a measure of the carbon dioxide content. Other indicatorssuch as phenol sulfone phthalein can be used in a similar manner.

Example I V.-Pr0tein in blood or plasma In this case a cellulose acetatemicro pore tyne of tape is suitable. A similar tape can be made fromTeflon, polyethylene, cellophane, cellulose nitrate, styrene orpolyvinylchloride polymers. In this case the pores are of a diameterwhich allow plasma protein molecules to pass but not red blood cells.

The whole blood or plasma is placed on the porous tape Which is aboveand touching the test tape. The test tape is melted with an alkalinebuffer at pH 10.5. After contact for 5 minutes the top tape is lifted.The test tape is now sprayed with a dilute solution of phosphotungsticacid. The test tape now goes through the heating zone at 80 C. for 5minutes and then dried at C. The blue spots are read on the densitometerusing a green filter for the protein content.

Example V.Preparing the bonded tape The bonded tape may be made bymaking a dilute solution of cellulose acetate in ethylene dichloride.The solution is sprayed evenly and lightly on one side of a tape made ofWhatman #3 filter paper as a sample paper. On drying a film of porouscellulose acetate covers one side of the paper. A dilute solution ofcellulose nitrate or a mixture of cellulose nitrate and celluloseacetate in methyl Cellosolve may be used in the same way or by paintingin a thin film (of the order of 0.1 mil in thickness) on one side of thepaper. If carefully done the test paper does not lose its absorbentqualities.

In another procedure a film of cellulose acetate, slightly wider thanthe porous test tape is fed into a pressing device so that the poroustape covers one side of the test tape completely and folds over theedges of the test tape. Applying heat and pressure a bond is formed sothat a bonded tape is available with a porous tape bonded to a test tape(in one case cited Whatman #3 paper).

Although the present invention has been described in conjunction withpreferred embodiments, it is to be understood that modifications andvariations may be resorted to without departing from the spirit andscope of the invention as those skilled in the art will readilyunderstand. Such modifications and variations are considered to bewithin the purview and scope of the invention and appended claims.

I claim:

1. A device for the automatic analysis of fluids for their chemicalcomponents, comprising in combination: means for dispensing a liquidsample in the form of a drop on a tape combination, said tapecombination including a transparent porous portion travelling with andin close contact with an absorbent tape, said absorbent tape having beenimpregnated with a reagent for producing a color reaction with thecomponent of the sample sought, a transfer station maintained atconstant temperature through which the drop of sample supported on saidporous tape passes and in which station said drop and said tapes movesfor a fixed period of time, the component for assay passing through saidporous tape to said reagent containing absorbent tape, a washing stationwhere water flows over the porous tape to remove the spent sample, adrying station where warm dry air dries the tape combination and areadout station where a beam of collimated monochromatic light passesthrough said tape combination and a sensing device to a recorder forrecording the light characteristics.

2. A device as claimed in claim 1, wherein said means for dispensing aliquid sample includes a dispensing station including means thereat tohold at least one capillary tube in the horizontal position and tiltingmeans to tilt said capillary tube to the substantially vertical positionso as to dispense a drop on said tape combination.

3. A device for the automatic chemical analysis of fluids for theircomponents, comprising, in combination: means for dispensing a liquidsample in the form of a drop on a tape combination, said tapecombination including a porous portion travelling with and in closecontact with an absorbent tape, means for continously irnpregnating saidabsorbent tape with a reagent for producing a color reaction with thecomponent of the sample sought, a transfer station maintained atconstant temperature through which the drop of sample supported and saidporous tapes moves for a fixed period of time, the component for assaydiffusing through said porous tape to said reagent containing absorbenttape, a separation station where the porous tape is removed from theabsorbent tape, and a drying station whereat warm dry air blows over theabsorbent tape, said absorbent tape then passing through a densitoineterwhere the intensity of color produced is evaluated by means of acombination of a collimated light source, a photo sensing device and asignal recorder.

4. A device as claimed in claim 3, wherein said means for dispensing aliquid sample includes a dispensing station including means thereat tohold at least one capillary tube in the horizontal position and tiltingmeans to tilt said capillary tube to the vertical position so as todispense a drop on said tape combination.

References Cited UNITED STATES PATENTS 3,036,893 5/1962 Natelson 23-2303,092,465 6/1963 Adams et al. 23--253 3,261,668 7/1966 Natelson 23-253JAMES H. TAYMAN, IR., Primary Examiner.

